Testicular hyperechogenic foci (THF) are associated with Klinefelter's syndrome, cryptorchidism, infertility, and testicular germ cell neoplasia. The aims of the study were to evaluate THF in relation to etiology of azoospermia and to Sertoli cell dysfunction. The structures inside the scrotum of consecutive non-vasectomized, azoospermic were examined by ultrasonography, and hormone (FSH, LH, testosterone, and prolactin), and genetic analyses (karyotype, Y microdeletions, and CFTR mutations) were performed. At testicular ultrasonography, patients were graduated into: pronounced THF (>7 THF per transducer field), distributed universally (uTHF) or collected in plaques (pTHF), borderline THF (bTHF; 3-7 THF per transducer field), or no THF (<3 THF per transducer field). Diagnostic testicular biopsy was taken open or with TruCut needle (14G). THF status was sufficiently described in 382 of 449 potential participants, and testicular histology was available in 300 cases. Presence of ultrasonographically detectable THF was compared to presence of testicular microlithiasis (TM) detected histologically. Sertoli cell dysfunction was investigated in a subgroup using a three-stage immunoperoxidase technique for detection of cytokeratin-18 (CK-18). The prevalence of THF was 13.4%. uTHF was found in 11 men (2.9%), the pattern was bilateral in four while other four had bTHF in the other testis. pTHF was detected in eight cases (2.1%), and except for one case with Klinefelter's syndrome, pTHF was in all cases occurring unilaterally. bTHF was detected in 32 cases (8.4%), bilaterally in 17 (53%). Pronounced THF was significantly associated with testicular malignancy. CK-18 was detected in more azoospermic men with sperm production in ≤50% seminiferous tubules than in azoospermic men with spermatogenesis in ≥90% of seminiferous tubules and normal controls (p < 0.05). Unfortunately, TM detected histologically was not detected in any patient expressing THF, and neither THF nor TM was detected in any of the patients examined for CK-18. Sertoli cell dysfunction was not associated with testicular microlithiasis or hyperechogenic foci.